in vivo 21: 813-818 (2007)

Down-regulation of 4 in Human Placenta Throughout Pregnancy

MARIA DE FALCO1*, LUIGI COBELLIS2*, MARCO TORELLA2, GENNARO ACONE2, LAURA VARANO3, ANNA SELLITTI1, ANGELO RAGUCCI2, GABRIELE COPPOLA4, ROBERTO CASSANDRO5, VINCENZA LAFORGIA1, LORENZO VARANO1 and ANTONIO DE LUCA4

1Department of Biological Sciences, Section of Evolutionary and Comparative Biology, University of Naples "Federico II", Naples; Departments of 2Gynecology, Obstetric and Reproductive Science and 4Medicine and Public Health, Section of Clinical Anatomy, Second University of Naples, Naples; 3IRCCS "Casa Sollievo della Sofferenza" Hospital, San Giovanni Rotondo (Foggia); 5San Giuseppe Hospital, Service of Pneumology, Milan, Italy

Abstract. Background: The family of mammalian Over the past decade, a family of related water-transporting (AQP) consists of 12 known members, each with a specific called aquaporins (AQP) has been identified (1-3). tissue distribution and membrane localization pattern. AQP4 They are a family of channel-forming transmembrane is the first member of this family identified in biological proteins that facilitate the movement of water, glycerol and membranes. This water channel is primarily expressed other solutes across the plasma membrane of cells (4-8). The in astrocytes but is also localized in ependymocytes and family of mammalian AQPs consists of 12 known members, endothelial cells, suggesting its involvement in the movement each with a specific tissue distribution and membrane of water between the blood and brain, and between the brain localization pattern (9), but many more from amphibians, and cerebrospinal fluid (CSF). To date, the regulation of plants, yeast, bacteria and other lower organisms have been AQP4 expression in the human placenta has not been studied. cloned (3). AQPs are small (~30 kDa) hydrophobic proteins The purpose of this work was to investigate AQP4 localization that assemble in membranes as tetramers. The aquaporins and expression in the human placenta during gestation. can be classified functionally into two groups: AQP1, AQP2, Materials and Methods: A total of 30 samples, 15 full-term AQP4, AQP5 and AQP8 are mainly permeable to water; placentae and 15 chorionic villous samples from first trimester, AQP3, AQP6, AQP7 and AQP9 are also water permeable, for the immunohistochemical analysis of AQP4 expression but in addition they have significant permeability to small were used. The gestation period ranged from 5 to 40 weeks. solutes such as glycerol (AQP3, AQP7 [aquaglyceroporins]) Results: A decrease of AQP4 expression in the and larger neutral solutes (AQP9), and, possibly under some syncytiotiophoblast from the first to the third trimester of conditions, to ions (AQP6). The sites of aquaporin gestation, in contrast with an increased expression shown by expression suggest a role in renal water reabsorption, endothelial cells and stroma of placental villi was found. cerebrospinal fluid (CSF) dynamics, aqueous fluid dynamics, Conclusion: Our results may suggest that AQP4-mediated glandular secretion and other physiological processes (3). maternal-fetal fluid exchange could play an important role in AQP4 was the first aquaporin to be observed and the control of ion homeostasis and water balance in the identified in biological membranes (9). AQP4 was initially human placenta throughout pregnancy. cloned from rat lung (10); subsequently isoforms from rat brain (11) and ortholog from human (12) and mouse (13) were sequenced (3). AQP4 is the most abundant water channel in brain, where it is mainly localized in *Both authors contributed equally to this study. pericapillary astrocyte foot processes, glial limiting membranes (comprising the outer brain surface and Correspondence to: Dr. Antonio De Luca, Department of Medicine subependyma) and ependyma (14-16). Recently, AQP4 and Public Health, Section of Clinical Anatomy, Second University of Naples, Via L. Armanni 5, 80138 Naples, Italy. Fax: +39 081 was also found in the brain vascular endothelium (8, 10, 458225, e-mail: [email protected] 11, 14, 17). This distribution suggests the involvement of AQP4 in the movement of water between the blood and Key Words: Aquaporin 4, placenta, pregnancy. brain and between the brain and CSF. Recent phenotype

0258-851X/2007 $2.00+.40 813 in vivo 21: 813-818 (2007) studies in transgenic mice lacking AQPs 1-5 have been Laboratories) was used as the chromogen and hematoxylin was very informative (18, 19). For example, mice lacking AQP4 used as nuclear counterstain. Negative controls for each tissue have impaired hearing (3, 20). The phenotype studies section were prepared by substituting the primary antiserum with non-immune IgG. For each experiment, all slides were stained in suggest that aquaporins are important for rapid, near- a single batch and thus received equal staining. isosmolar fluid transport, as occurs in kidney proximal Immunohistochemical staining intensity was evaluated by ranking: tubules and salivary glands, and for water transport when it 0 (absent), 1 (weak), 2 (moderate) or 3 (intense) as described by is driven by established osmotic gradients, as in kidney Seelam et al. (27). For each specimen, an HSCORE value was collecting ducts. The phenotype studies have also shown derived by summing the percentages of cells/areas that stained at that the tissue-specific expression of an aquaporin does not each intensity and multiplying that by the weighted intensity of the indicate physiological significance – for example, deletion staining. For example: HSCORE = ™ Pi (i + 1) where i represents the intensity scores and P the corresponding percentage of of AQP4 in skeletal muscle and gastric parietal cells does i cells/areas. not impair skeletal muscle function (21), or gastric acid An average of 22 fields were observed for each tissue by three production (3, 22). observers at different times and the average score was used. All Pregnancy has been shown to up-regulate aquaporin values were expressed as mean ± standard error of mean (SEM) expression in other organs such as the uterus (8, 23, 24), but and differences were compared using Student’s t-test. the regulation of AQP4 expression in the human placenta during pregnancy has not been studied. The placenta is Results composed of maternal and fetal material and plays an integral role not only in the supply of nutrients to the fetus Distribution of AQP4 in human placenta during gestation. The but also in the maintenance of pregnancy (25). The human localization and distribution of AQP4 in human placenta placenta is able to synthesize and secrete a variety of during gestation by immunohistochemistry was investigated. hormones and molecules that regulate the fine balance We observed that in the first trimester, AQP4 was localized between proliferation, differentiation and invasion of in the cytoplasm of syncytiotrophoblast at an intense level of trophoblastic cells. In order to better clarify AQP4 expression (Figure 1a). In contrast, the AQP4 expression in localization in human placenta, an immunohistochemical the cytotrophoblast was weak. In addition, we observed a analysis of AQP4 expression throughout pregnancy in moderate immunopositivity for AQP4 in the stroma of first physiological conditions was carried out. trimester placental villi and in the cytoplasm of endothelial cells (Figure 1b). During the third trimester of gestation, Materials and Methods AQP4 immunopositivity slightly decreased in the syncytiotrophoblast (Figure 1c), while AQP4 immunopositivity Samples. Human placental samples were obtained with informed increased in the stroma of placental villi and in the cytoplasm consent from patients undergoing surgery such as cesarean section of endothelial cells (Figure 1c). The AQP4 immunopositivity for normal placenta and uterine evacuation for normal chorionic in the cytotrophoblast was always weak during the third villi. A total of 30 samples, 15 full-term placentae and 15 chorionic trimester of gestation (Figure 1d). villous samples from the first trimester were used and the gestation In Figure 2 the expression pattern of AQP4 during period ranged from 5 to 40 weeks. The specimens were immediately fixed in formalin for immunohistochemistry. pregnancy, as detected by immunohistochemical staining intensity analysis, is depicted. We observed a decrease of Immunohistochemistry. Immunohistochemistry was carried out AQP4 expression from the first to the third trimester of essentially as described previously (26). Briefly, sections embedded gestation in the syncytiotrophoblast, in contrast with an in paraffin from each specimen were cut at 5 Ìm, mounted on glass increased expression of AQP4 in endothelial cells and and dried overnight at 37ÆC. All sections were then deparaffinized stroma of placental villi. The AQP4 expression in the in xylene, rehydrated through a graded series of alcohol and cytotrophoblast appeared unchanged from the first to the washed in phosphate-buffered saline (PBS). PBS was used for all subsequent washes and for antiserum dilution. Tissue sections were third trimester of gestation. quenched sequentially in 3% hydrogen peroxide and blocked with PBS-6% non-fat dry milk (Bio-Rad, Hercules, CA, USA) for 1 h Discussion at room temperature. Slides were then incubated at 4ÆC overnight with rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa AQP4 is expressed in astrocyte foot processes near blood Cruz, CA, USA) raised against AQP-4 (sc-6014) at a 1:400 dilution. vessels in rat (14, 15) and human (28) brain, as well as in After several washes (3x5 min) to remove excess antibody, the ependymal and pial surfaces in contact with CSF (15, 29, slides were incubated with diluted anti-goat biotinylated antibody, anti-rabbit biotinylated antibody or anti-mouse biotinylated 30). This localization suggests that AQP4 plays a critical antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h. All role in brain water balance (3). AQP4 appears to be the slides were then processed by the ABC method (Vector selective for the passage of water and is unique among the Laboratories) for 30 min at room temperature. Novared (Vector aquaporins in its exceptionally high intrinsic water

814 De Falco et al: AQP4 Localization in Human Placenta

Figure 1. Localization of aquaporin 4 (AQP4) in human placenta throughout gestation. a) AQP4 localization in the first trimester of gestation showing the high immunopositivity in the syncytiotrophoblast, x150; b) First trimester placental villous showing the intense immunopositivity of AQP4 in the syncytiotrophoblast and the moderate positivity in the cytoplasm of endothelial cells (arrows), x640; c) AQP4 localization in the third trimester of gestation, x150; d) AQP4 moderate immunolocalization in the syncytiotrophoblast, stroma and endothelial cells of the third trimester placental villi, x300; e) Representative negative control of a placenta of the first trimester of gestation, x100; f) Representative negative control of a placenta of the third trimester of gestation, x100.

815 in vivo 21: 813-818 (2007)

AQP4 expression pattern in human placenta during gestation seems to suggest an important role of this protein in the regulation of maternal-fetal fluid exchange between placental cells and capillaries. In addition, AQP4 could play a key role in the regulation of amniotic fluid volume homeostasis. In conclusion, since there is a requirement for increased fluid transfer to the conceptus, the expression of AQP4 in human placenta throughout pregnancy seems to be important to maintain fetal growth.

References

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